Genomic Landscape of RUNX1-Familial Platelet Disorder with Myeloid Malignancies Reveals Rising Clonal Hematopoiesis

Abstract Familial platelet disorder with associated myeloid malignancies (FPDMM) is a rare autosomal dominant disease caused by germline RUNX1 mutations. FPDMM patients have defective megakaryocytic development, low platelet counts, prolonged bleeding times, and a life-long risk (20-50%) of developing hematological malignancies. FPDMM is a rare genetic disease in need of comprehensive clinical and genomic studies. In early 2019 we launched a longitudinal natural history study of patients with FPDMM at the NIH Clinical Center and by May 2021 we have enrolled 98 patients and 100 family controls from 55 unrelated families. Genomic data have been generated from 56 patients in 24 families, including whole exome sequencing (WES), RNA-seq, and single-nucleotide polymorphism (SNP) array. We have identified 21 different germline RUNX1 variants among these 24 families, which include lost-of-function mutations throughout the RUNX1 gene, but pathogenic/likely pathogenic missense mutations are mostly clustered in the runt-homology domain (RHD). As an important form of RUNX1 germline mutations, five splice site variants located between exon 4-5 and exon 5-6 were identified in 6 families, which led to the productions of novel transcript forms that are predicted to generate truncated RUNX1 proteins. Large deletions affecting the RUNX1 gene are also common, ranging from 50 Kb to 1.5Mb, which were detected in 8 of the 55 enrolled families. Besides RUNX1, copy number variation (CNV) analysis from both SNP array and WES showed limited CNV events in non-malignant FPDMM patients. In addition, fusion gene analysis did not detect any in-frame fusion gene in these patients, indicating a relatively stable chromosome status in FPDMM patients. Somatic mutation landscape shows that the overall mutation burden in non-malignant FPDMM patients is lower than AML or other cancer types. However, in 13 of the 44 non-malignant patients (30%), somatic mutations were detected in at least one of the reported clonal hematopoiesis of indeterminate potential (CHIP) genes, significantly higher than the general population (4.3%). Moreover, 85% of our patients who carried CHIP mutations are under 65 years of age; in the general population, only 10% of people above 65 years of age and 1% of people under 50 were reported to carry CHIP mutations. Among mutated genes related to clonal hematopoiesis, BCOR is the most frequently mutated gene (5/44) in our FPDMM cohort, which is not a common CHIP gene among the general population. Mutations in known CHIP genes including SF3B1, TET2, and DNMT3A were also found in more than one patient. In addition, sequencing of 5 patients who already developed myeloid malignancies detected somatic mutations in BCOR, TET2, NRAS, KRAS, CTCF, KMT2D, PHF6, and SUZ12. Besides reported CHIP genes or leukemia driver genes, 3 unrelated patients carried somatic mutations in the NFE2 gene, which is essential for regulating erythroid and megakaryocytic maturation and differentiation. Two of the NFE2 mutations are nonsense mutations, and the other is a missense mutation in the important functional domain. NFE2 somatic mutations may play important roles in developing malignancy because 2 of the 3 patients already developed myeloid malignancies. For multiple patients in our cohort, we have sequenced their DNA on multiple timepoints. We have observed patients with expanding clones carrying FKBP8, BCOR or FOXP1 mutations. We have also observed a patient with relatively stable clone(s) with somatic BCOR, DNMT3A, and RUNX1T1, who have been sampled over more than four years. We will follow these somatic mutations through sequencing longitudinally and correlate the findings with clinical observations to see if the dynamic changes of CHIP clones harboring the mutations give rise to MDS or leukemia. In summary, the genomic analysis of our new natural history study demonstrated diverse types of germline RUNX1 mutations and high frequency of somatic mutations related to clonal hematopoiesis in FPDMM patients. These findings indicate that monitoring the dynamic changes of these CHIP mutations prospectively will benefit patients' clinical management and help us understand possible mechanisms for the progression from FPDMM to myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

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A Novel Inherited Single-Nucleotide Mutation in 5′-UTR in the Transcription Factor RUNX1 in Familial Platelet Disorder with Propensity To Develop Myeloid Malignancies.

Blood ◽

10.1182/blood.v108.11.1917.1917 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1917-1917 ◽

Cited By ~ 2

Author(s):

Keita Kirito ◽

Toru Mitsumori ◽

Takahiro Nagashima ◽

Masae Kunitama ◽

Kei Nakajima ◽

...

Keyword(s):

Transcription Factor ◽

Point Mutation ◽

Aberrant Expression ◽

Single Nucleotide ◽

Myeloid Malignancies ◽

Single Nucleotide Mutation ◽

Runx1 Gene ◽

Nucleotide Mutation ◽

Platelet Disorder ◽

Familial Platelet Disorder

Abstract RUNX1 transcription factor plays pivotal roles in the development of definitive hematopoiesis. Allelic loss of the gene causes complete absence of fetal liver hematopoiesis. In addition to normal hematopoiesis, aberrant expression of RUNX1 is also involved in the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Familial platelet disorder with propensity to develop myeloid malignancies (FPD/AML, OMIM 601399) is a rare autosomal dominant disorder characterized by thrombocytopenia, dysfunction of platelets and predisposition to the development of myeloid malignancies. Recent studies revealed that inherited mutation of RUNX1 gene is responsible for the onset of FPD/AML. To date, 12 families of FPD/AML have been reported in the literature, and point mutation in the RUNT domain or loss of heterozygocity (LOH) of the gene has been identified in the pedigree. Here, we report a Japanese family with FPD/AML with a novel mutation of RUNX1 gene. A 38-year-old man was admitted to our hospital because of MDS (RAEB) in August 2003. Cytogenetic analysis revealed abnormal karyotype; 46XY, t (7; 8)(q34; q11). In addition, prolongation of bleeding time and abnormal platelet aggregation were observed. His son and daughter also showed mild bleeding tendency and had mild thrombocytopenia. In April 2006, the daughter developed MDS (RAEB) with trisomy 8 at age 16. After informed consent, blood samples were obtained from all family members and all 9 exons of RUNX1 gene were sequenced. We identified a novel G to T single-nucleotide mutation in the 5′-untranslated region (5′-UTR) in the exon1, corresponding to position 102 of RUNX1 transcripts (NCBI accession no. D43969). This mutation was also found in all the affected individuals but not in the healthy members. To investigate the possibility of hemizygous intragenic deletion of the gene, we performed an array- based comparative genomic hybridization using Affymetrix GeneChip Human Mapping 250K set including 23 SNPs in RUNX1 gene. We found no loss of heterozygosity of RUNX1 gene in the affected members. Because the mutation is located in 5′-UTR, we investigated whether this mutation might affect the expression of RUNX1 transcripts. Transcription of RUNX1 is regulated by two distinct promoter regions, distal and proximal, resulting in the generation of transcripts having different 5′-UTRs. The 5′-UTR of transcripts controlled by distal promoter contains exon1 (distal form), whereas that of transcripts controlled by proximal promoter contains exon3 but not exon1 (proximal form). We analyzed the expression level of both transcripts from bone marrow cells using quantitative RT-PCR. Affected individuals showed 10 to 15 times higher expression of the distal form of RUNX1 transcripts, compared to normal controls (n=3), MDS patients (n=3) and AML patient (n=1). Considering that not only haploinsufficiney but also overexpression of RUNX1 can cause AML, aberrant expression of RUNX1 induced by the point mutation in 5′-UTR may be involved in progression of FPD/AML.

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RUNX1-mutated families show phenotype heterogeneity and a somatic mutation profile unique to germline predisposed AML

Blood Advances ◽

10.1182/bloodadvances.2019000901 ◽

2020 ◽

Vol 4 (6) ◽

pp. 1131-1144 ◽

Cited By ~ 8

Author(s):

Anna L. Brown ◽

Peer Arts ◽

Catherine L. Carmichael ◽

Milena Babic ◽

Julia Dobbins ◽

...

Keyword(s):

Somatic Mutations ◽

Published Data ◽

Data Sets ◽

Gene Deletions ◽

Phenotypic Differences ◽

Recurrent Mutations ◽

Platelet Disorder ◽

Related Transcription Factor ◽

Phenotype Heterogeneity ◽

Familial Platelet Disorder

Abstract First reported in 1999, germline runt-related transcription factor 1 (RUNX1) mutations are a well-established cause of familial platelet disorder with predisposition to myeloid malignancy (FPD-MM). We present the clinical phenotypes and genetic mutations detected in 10 novel RUNX1-mutated FPD-MM families. Genomic analyses on these families detected 2 partial gene deletions, 3 novel mutations, and 5 recurrent mutations as the germline RUNX1 alterations leading to FPD-MM. Combining genomic data from the families reported herein with aggregated published data sets resulted in 130 germline RUNX1 families, which allowed us to investigate whether specific germline mutation characteristics (type, location) could explain the large phenotypic heterogeneity between patients with familial platelet disorder and different HMs. Comparing the somatic mutational signatures between the available familial (n = 35) and published sporadic (n = 137) RUNX1-mutated AML patients showed enrichment for somatic mutations affecting the second RUNX1 allele and GATA2. Conversely, we observed a decreased number of somatic mutations affecting NRAS, SRSF2, and DNMT3A and the collective genes associated with CHIP and epigenetic regulation. This is the largest aggregation and analysis of germline RUNX1 mutations performed to date, providing a unique opportunity to examine the factors underlying phenotypic differences and disease progression from FPD to MM.

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Clonal Hematopoiesis in Patients with Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2020-139652 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 25-26

Author(s):

Michelle Nash ◽

Adrianna Vlachos ◽

Marcin W. Wlodarski ◽

Jeffrey Michael Lipton

Keyword(s):

General Population ◽

Myelodysplastic Syndrome ◽

Somatic Mutation ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Age Of Onset ◽

Bone Marrow Failure ◽

Large Subunit ◽

Diamond Blackfan Anemia ◽

Clonal Hematopoiesis

Background: Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by anemia, congenital anomalies and a predisposition to cancer. Patients usually present during infancy or early childhood, but can also be diagnosed as adults. In the vast majority of cases DBA is due to a mutation in a gene encoding a small or large subunit-associated ribosomal protein (RP) leading to RP haploinsufficiency. In a study of 702 patients enrolled in the DBA Registry (DBAR), the observed to expected ratio for acute myeloid leukemia (AML) was 28.8 and for myelodysplastic syndrome (MDS), 352.1 (Vlachos et al, Blood, 2018). The average age of onset for MDS in the DBA cohort was 26 years, compared to 60-70 years in the general population. Evolving clonal hematopoiesis (CH) with age has been observed as a precursor to MDS, with CH rarely observed in individuals younger than 40 years of age. Thus we hypothesized that the young age at the development of MDS in DBA would be presaged by evolving CH. Objective: The primary objective was to perform whole exome sequencing (WES) specifically screening for previously reported somatic mutations in 56 genes associated with CH (Jaiswal et al, NEJM, 2014). Design/Method: A total of 69 samples were analyzed from 65 patients, mostly targeting patients older than 18 years (median age 30 years). Multiple samples were run on patients who had available samples in the DBAR Biorepository to determine rate of acquisition of mutations. 468 age- and sex-matched healthy controls were made available from GeneDx who performed the WES for the study. We used a threshold for variant calling of minimum 5% with a minimum of 2 variant reads. Results: Three of the 65 DBA patients (5%) were found to have somatic mutations in STAG1, U2AF1, SF3B1, and DNMT3A at 8, 20, 41, and 70 years, respectively (Table 1). The patient who was 20 years of age had a sample in the DBAR biorepository from when he was age 8 years which was found to have a different somatic mutation (STAG1) than was found at present (U2AF1). This patient did go on to develop MDS at the age of 21 years. In comparison, of the 468 controls, 4 (0.8 %) had a somatic mutation in SF3B1, LUC7L2, DNMT3A, and LUC7L2 at ages 12, 31, 33 and 40 years, respectively. Conclusion: Patients with DBA show more somatic mutations as compared to controls (p<0.05). This early acquisition of mutations may be the driving force for their developing MDS at an earlier age than that of the general population. Further studies with more sensitive methods are warranted to accurately determine the prevalence of somatic CH mutations and their potential association with the development of myelodysplastic syndrome in these patients. Disclosures No relevant conflicts of interest to declare.

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Down-regulation of the RUNX1-target gene NR4A3 contributes to hematopoiesis deregulation in familial platelet disorder/acute myelogenous leukemia

Blood ◽

10.1182/blood-2010-12-325555 ◽

2011 ◽

Vol 118 (24) ◽

pp. 6310-6320 ◽

Cited By ~ 30

Author(s):

Dominique Bluteau ◽

Laure Gilles ◽

Morgane Hilpert ◽

Iléana Antony-Debré ◽

Chloe James ◽

...

Keyword(s):

Regulatory Gene ◽

Dominant Negative ◽

Myelogenous Leukemia ◽

Down Regulation ◽

Α Subunit ◽

Runx1 Gene ◽

Platelet Disorder ◽

Clonogenic Potential ◽

Acute Myelogenous ◽

Familial Platelet Disorder

Abstract RUNX1 encodes a DNA-binding α subunit of the core-binding factor, a heterodimeric transcription factor. RUNX1 is a master regulatory gene in hematopoiesis and its disruption is one of the most common aberrations in acute leukemia. Inactivating or dominant-negative mutations in the RUNX1 gene have been also identified in pedigrees of familial platelet disorders with a variable propensity to develop acute myeloid leukemia (FPD/AML). We performed analysis of hematopoiesis from 2 FPD/AML pedigrees with 2 distinct RUNX1 germline mutations, that is, the R139X in a pedigree without AML and the R174Q mutation in a pedigree with AML. Both mutations induced a marked increase in the clonogenic potential of immature CD34+CD38− progenitors, with some self-renewal capacities observed only for R174Q mutation. This increased proliferation correlated with reduction in the expression of NR4A3, a gene previously implicated in leukemia development. We demonstrated that NR4A3 was a direct target of RUNX1 and that restoration of NR4A3 expression partially reduced the clonogenic potential of patient progenitors. We propose that the down-regulation of NR4A3 in RUNX1-mutated hematopoietic progenitors leads to an increase in the pool of cells susceptible to be hit by secondary leukemic genetic events.

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Whole Exome Sequencing Detecting Kinesin Family Gene Defects In Myeloid Neoplasm

Blood ◽

10.1182/blood.v122.21.2762.2762 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2762-2762

Author(s):

Chantana Polprasert ◽

Hideki Makishima ◽

Bartlomiej P Przychodzen ◽

Naoko Hosono ◽

Wenyi Shen ◽

...

Keyword(s):

Research Funding ◽

Somatic Mutations ◽

Snp Array ◽

Chromosome 17 ◽

Therapeutic Window ◽

Myeloid Malignancies ◽

Myeloid Neoplasm ◽

Myeloid Neoplasms ◽

Whole Exome ◽

Kinesin Family

Abstract Clinical and pathomorphologic diversity in MDS is a reflection of heterogeneity of molecular lesions. Somatic mutations and chromosomal deletions/amplifications affect various pathways in a convergent and divergent fashion, generate phenocopy and can occur in a variety of combinations. Recent technological advances, including high density arrays and the new generation sequencing (NGS) led to the discovery of novel pathway mutations or gene families affected by somatic defects, e.g., cohesin or spliceosomal mutations. We have performed whole exome NGS of paired (tumor/germ line) samples in 222 patients with myeloid neoplasms from the Cleveland Clinic and University of Tokyo. Clinical parameters were studied including age, gender, overall survival (OS), bone marrow blast count, and metaphase cytogenetics. Additionally, we also used in our analysis data sets from 197 AML included in the Cancer Genome Atlas (TCGA). We found 1.4% (6/419) of non-canonical somatic mutations of KIF2Bwhich is a member of kinesin13 family located on the long arm of chromosome 17; 3 cases from our cohort (p.V32M (c.G94A), p.T113M (c.C338T), p.R163C (c.C487T)) and 3 cases from TCGA database (p.T47M (c.C140T), p.T310M (c.C929T), p.H551N (c.C1651A)). By analyzing clonal architecture and intra-tumor heterogeneity in 2 cases (RCMD and RAEB) by targeted deep sequencing, allelic frequencies of KIF2B mutations were more than 45% and larger than for any other concomitant mutations, suggesting that KIF2B mutations might consequently constitute ancestral events followed by subclonal acquisitions of the other mutations. Of note is that 6 non-sense mutations were also reported in lung cancer. Based on SNP-array mapping of chromosomal abnormalities, deletions of 17q involving the KIF2B locus (17q22) was present about 3% (6/215) of myeloid neoplasm. KIF2B defects were frequently detected in higher-risk MDS and AML phenotypes (9%). KIF2B performed an important role in regulation of kinetochore-microtubule attachment. Previous studies showed that the velocity of chromosomes’ movement in KIF2B-deficient cells is reduced 80% comparing to control and fail to perform cytokinesis. In our series, 56% of myeloid neoplasms with KI2B defects had complex cytogenetics and 67% cases of them were also UPD, suggesting that KIF2B defects might lead to inducing abnormal chromosomal movements and segregations. We then, expanded our study to the whole kinesin gene family: 17 somatic mutations and 57 deletions were identified in KIF1A (n=6), KIF23 (n=1), KIF26A (n=1), KIF27 (n=7), KIF1C (n=9), KIF21B (n=2), KIF13A (n=10), KIF14 (n=2), KIF17 (n=15), KIF25 (n=1), KIF3C (n=8), KIF6 (n=2) and CENPE (n=10). All mutations were heterozygous and mutually exclusive. By survival analysis of such mutated cases, a tendency towards worse prognosis was observed (HR; 1.72, 95%CI 0.86-3.37). Analysis of concomitant mutations associated with whole kinesin family mutations or deletions showed that most frequently affected genes are TET2 (n=14), DNMT3A (n=8), IDH1/2 (n=8) and MLL (n=5), all involved in epigenetic regulation. In conclusion, somatic mutations in kinesin family genes are found in myeloid malignancies and might be responsible for another pathogenesis of the disease. KIF2B is most frequently found in myeloid malignancies and associated with aggressive type of MDS. Since knockout mice of multiple kinesin family genes (KIF5A, KIF16B and EG5) were lethal in embryo and all the mutations occur in a heterozygous configuration, it is likely synthetic lethal approach might create therapeutic window between defective malignant cells and healthy controls. Kinesin family of motor proteins may be an emerging novel therapeutic target. In fact some kinesins have been already successfully targeted in solid tumors. Disclosures: Polprasert: MDS foundation: Research Funding. Makishima:AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

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Functional classification of RUNX1 variants in familial platelet disorder with associated myeloid malignancies

Leukemia ◽

10.1038/s41375-021-01200-w ◽

2021 ◽

Author(s):

Melanie Decker ◽

Tim Lammens ◽

Alina Ferster ◽

Miriam Erlacher ◽

Ayami Yoshimi ◽

...

Keyword(s):

Functional Classification ◽

Myeloid Malignancies ◽

Platelet Disorder ◽

Familial Platelet Disorder

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Unraveling a Novel Mechanism of RUNX1 Activity in Familial Platelet Disorder with Associated Myeloid Malignancy

Blood ◽

10.1182/blood.v128.22.2693.2693 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2693-2693

Author(s):

Halah I. Alkadi ◽

Tatiana S. Karpova ◽

Erika Mijin Kwon ◽

Lisa J. Garrett ◽

Yongxing Gao ◽

...

Keyword(s):

Critical Role ◽

Hek293 Cells ◽

Luciferase Assay ◽

Binding Motif ◽

Myeloid Malignancy ◽

C Terminus ◽

Runx1 Gene ◽

Platelet Disorder ◽

Fret Efficiency ◽

Familial Platelet Disorder

Abstract Acute myeloid leukemia (AML) has a higher incidence and death rate than all other types of adult-onset acute leukemia in the USA, thus requiring a better understanding of the molecular mechanisms behind its progression. Since familial platelet disorder with associated myeloid malignancy (FPDMM) is closely related to AML and is caused by mutations in the RUNX1 gene, elucidation of RUNX1 in the development of FPDMM serves as a model for understanding the genesis of AML. FPDMM is a rare autosomal dominant disorder. FPDMM patients are characterized with defective megakaryopoiesis, abnormal platelet count and function, and bleeding disorders. Importantly, ~60% of patients develop hematological malignancies later on in their lives. FPDMM patients carry heterozygous, germline mutations in the RUNX1 gene. RUNX1 is a transcription factor that plays a critical role during early stages of definitive hematopoiesis, and megakaryopoiesis. Significantly, RUNX1 mutations have been reported in many cases of AML and myelodysplastic syndrome. RUNX1's C-terminal contains a VWRPY motif, which is a conserved binding site for transducin like enhancer of split1 (TLE1). TLE1 is a transcriptional corepressor that inhibits several transcription factors. Previous studies showed that RUNX1 missing the VWRPY motif could not bind TLE1, resulting in overexpression of RUNX1's target genes. However, the significance of RUNX1-TLE1 interaction was never investigated in regard to megakaryopoiesis, FPDMM pathogenesis, or leukemogenesis. Hence, there is a need to better understand the role of RUNX1-TLE1 interaction and their significance in megakaryopoiesis in general. A new FPDMM family has been identified carrying a GC insertion at the end of RUNX1's C-terminus. Genomic DNA sequencing of two patients from the family confirmed the mutation, which resulted in a frame shift mutation (L472fsX). As a result, the VWRPY motif is absent. Instead, the mutant protein contains additional, unrelated 123 amino acids, whose expression has been confirmed by western blot. Our hypothesis states that because the RUNX1 mutant lacks the TLE1 binding motif (VWRPY), its repression is defective which in turn affects normal megakaryopoiesis. Thereby, we are presenting a novel RUNX1 mutation in FPDMM and a possible novel mechanism that has never been studied before in FPDMM patients. To evaluate the effect of the mutation on RUNX1-TLE1 interaction, fluorescence resonance energy transfer (FRET) was performed in HEK293 cells. CFP-RUNX1 wild type (wt) and mutant co-transfected with YFP-CBFβ gave a FRET efficiency of 14% ± 2.5% and 16% ± 2.7%, respectively; suggesting that the mutation did not disrupt the physical binding between RUNX1 and its co-factor CBFβ. CFP-RUNX1 wt co- transfected with YFP-TLE1 gave an average of 10% ± 3.3% FRET efficiency, while CFP-RUNX1 mutant co-transfected with YFP-TLE1 gave an average of 0.65% ± 1.8% FRET efficiency, indicating no binding between the RUNX1 mutant and TLE1. These findings demonstrate that the existence of RUNX1's C-terminus mutation abolished RUNX1's interaction with TLE1. Furthermore, to assess the effect of the disrupted interaction between RUNX1 and TLE1 on RUNX1's activity, we performed a dual luciferase assay, which measures the promoter activity of a RUNX1's target, myeloid colony stimulating factor receptor (MCSFR). Results show that TLE1 was able to partially repress RUNX1 wt activity when co-transfected with CBFβ, consistent with previous data. On the contrary, TLE1 did not repress RUNX1 mutant activity, which resulted in increased RUNX1's target expression. Therefore, these preliminary results are consistent with the proposed regulatory role for RUNX1 and TLE1 during hematopoiesis. To corroborate these results, we have generated human induced pluripotent stem cells (iPSCs) from the FPDMM patient's blood cells containing the RUNX1 L472fsX mutation to model the defects in megakaryopoiesis. We are currently analyzing the hematopoietic differentiation of the mutated iPSCs and studying the mechanism through expression and pathway analysis of RNA-Seq data. Moreover, we have generated a mouse model closely representing the mutation using CRISPR-Cas9 system. Bothmodels will be used to provide a better understanding of megakaryopoiesis in general, and FPDMM pathogenesis and their progression to leukemia in particular. Disclosures No relevant conflicts of interest to declare.

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When are idiopathic and clonal cytopenias of unknown significance (ICUS or CCUS)?

Hematology ◽

10.1182/hematology.2021000272 ◽

2021 ◽

Vol 2021 (1) ◽

pp. 399-404

Author(s):

Afaf E. W. G. Osman

Keyword(s):

Somatic Mutations ◽

Genetic Mutations ◽

Sequencing Technology ◽

Myeloid Malignancies ◽

Clonal Hematopoiesis ◽

Exponential Increase ◽

Unknown Significance ◽

Risk Of Progression ◽

Clinical Consequences ◽

Increase In Risk

Abstract Rapid advances in sequencing technology have led to the identification of somatic mutations that predispose a significant subset of the aging population to myeloid malignancies. Recently recognized myeloid precursor conditions include clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of unknown significance (CCUS). These conditions can present diagnostic challenges and produce unwarranted anxiety in some instances. While the risk of progression to myeloid malignancies is very low in CHIP, true CCUS confers an exponential increase in risk. Idiopathic cytopenia of unknown significance (IDUS) lacks the predisposing genetic mutations and has a variable course. In this review we define the early myeloid precursor conditions and their risk of progression. We present our diagnostic approach to patients with unexplained cytopenias and discuss the clinical consequences of CHIP and CCUS.

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Familial Platelet Disorder and Predisposition to Myeloid Leukemia (FPD/AML) in the Absence of RUNX1 Mutations- A Report of Three Families.

Blood ◽

10.1182/blood.v114.22.4689.4689 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4689-4689

Author(s):

Jason N. Berman ◽

Stephen Couban ◽

Allan Ebbin ◽

Haydar Frangoul ◽

Maria B. Quisumbing ◽

...

Keyword(s):

Myeloid Leukemia ◽

Autosomal Dominant ◽

Conflicts Of Interest ◽

Gene Dosage ◽

Protein Product ◽

Dominant Negative ◽

Runx1 Gene ◽

Platelet Disorder ◽

Familial Platelet Disorder ◽

Dominant Negative Activity

Abstract Abstract 4689 Familial Platelet Disorder with Predisposition to Acute Myeloid Leukemia (FPD/AML) has been attributed to abnormalities of the RUNX1 gene, located at human chromosome 21q22. The protein product of this gene forms a critical component of the core binding factor (CBF) complex, which is required for normal hematopoiesis. Truncating mutations and deletions of RUNX1 (particularly in exons 3, 4, and 5, encoding the DNA binding and CBF-beta interaction domains) that result in haploinsufficiency or dominant-negative activity have been identified as the genetic mechanisms underlying FPD/AML. Clinically, these patients present with mild to moderate thrombocytopenia, platelet dysfunction, bleeding, and about a 35% risk of developing AML. We report on three unrelated families who share histories of autosomal dominant thrombocytopenia over two to five generations, bleeding, platelet aggregation defects, and development of myelodysplastic syndrome, AML or intriguingly, chronic myeloid leukemia (CML). Detailed genetic interrogation of the RUNX1 locus in the proband from each family, including complete sequencing of all 8 exons, flanking 50 base pair regions, and P1 and P2 promoters, as well as gene dosage studies, failed to demonstrate a causative lesion in the RUNX1 gene. Our findings strongly suggest that genetic loci other than RUNX1 are involved in some cases of autosomal dominant thrombocytopenia with a predisposition to both AML and CML. Identification of the disease-causing genes in these families will allow for prospective testing of family members, appropriate surveillance and early intervention in affected individuals, and potentially new molecular insights into leukemogenesis. We have broadened the genotype-phenotype correlation in FPD/AML beyond the RUNX1 gene and suggest that this syndrome may be more genetically heterogeneous than initially described. Disclosures: No relevant conflicts of interest to declare.

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Somatic PHF6 Mutations In Myeloid Malignancies

Blood ◽

10.1182/blood.v122.21.2514.2514 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2514-2514

Author(s):

Wenyi Shen ◽

Bartlomiej P Przychodzen ◽

Chantana Polprasert ◽

Naoko Hosono ◽

Brittney Dienes ◽

...

Keyword(s):

Deep Sequencing ◽

Research Funding ◽

Somatic Mutations ◽

Snp Array ◽

Illustrative Case ◽

Sequencing Analysis ◽

Data Set ◽

Myeloid Malignancies ◽

Dysmorphic Features ◽

Myeloid Neoplasms

Abstract X chromosome genomics is an important area of hematologic malignancy research because of frequent acquired X-abnormalities, location of important genes on this chromosome, and issues surrounding Lyonization (X-inactivation). For example, we previously described somatic mutations of UTX (KDM6A), a H3K27 demethylase located on chromosome Xp11.3, in aggressive myeloid neoplasms. In a companion abstract to the the results here, we also report loss of function somatic mutations of BRCC3 (Xq28), encoding a subunit of the BRCA1-BRCA2-containing complex. In an index young female case of a proliferative CMML with dysmorphic features, we have identified PHF6 mutation mosaisism (p.K44fs), confirmed by deep sequencing of bone marrow, skin and spleen tissues. Subsequently, we screened our MDS exome project data set, involving 206 patients with MDS and related neoplasms, and have detected and confirmed additional somatic PHF6mutations. Plant homeodomain finger protein 6 (PHF6) is a ubiquitously expressed 41 kDa protein that is conserved and vertebrate-specific. Human PHF6 is located on chrXq26.3. Germline mutations of PHF6 cause Borjeson−Forssman−Lehmann syndrome (BFLS), an X-linked mental retardation disorder characterized by truncal obesity, gynaecomastia, hypogonadism and other dysmorphic features. BFLS patients have been reported to develop leukemias. More recently, rare somatic PHF6mutations were detected in patients with T-ALL, but rarely also in AML. To assess the clinical associations and significance of PHF6 mutations, we analyzed NGS results in a total of 809 patients with MDS, MDS/MPN, MPN and AML. In addition we also investigated for the presence of PHF6 mutation in the TCGA AML data sets (n=199). All mutations in our patients were confirmed by Sanger sequencing and targeted deep NGS. In total, we identified 19/809 cases with PHF6 mutations; they were located throughout the gene including 15 SNVs and 4 indels. In addition TCGA pAML NGS results revealed PHF6 mutations in 6/199 cases, including 4 SNVs and 2 indels. Thus, PHF6 mutation occurs at a frequency of 2.5% in myeloid neoplasm and are most frequently observed in pAML (36%) together with sAML (32%) phenotypes. Gender distribution showed male predominance (84%), likely related to PHF6 locus on chrXq26.3. SNP-array karyotyping showed that deletions of Xq, involving PHF6locus (Xq26) were present in about 2% of myeloid neoplasms. Chromosome 7 abnormalities, including del(7q), were the most frequent lesions seen in conjunction with PHF6 mutations. Most commonly coinciding mutations were in RUNX1 (n=8), TET2 (n=4), ASXL1 (n=3) and U2AF1 (n=3) and unbiased statistical analysis confirmed the significant association between PHF6 and RUNX1 mutations (P=.002). Interestingly, all of 8 cases with concomitant RUNX1 and PHF6 mutations were diagnosed as high-risk diseases; 1 RAEB-2 and 7 AMLs. Deep sequencing analysis of 5 cases with coexisting PHF6 and RUNX1 mutations showed that PHF6 mutated clones were always significantly larger than RUNX1 mutated clones. Such a serial clonal acquisition pattern of ancestral PHF6 and secondary RUNX1 mutations was also observed clearly in an illustrative case with evolution from aplastic anemia (AA) to sAML, in which small clone of PHF6 was detected in AA sample and expanded during MDS stage, followed by secondary driver RUNX1 mutations at the stage. These findings suggest that RUNX1 mutations were acquired as a subclone of the main population with primary driver PHF6mutations. In conclusion, our results indicate that PHF6 mutations, as a recurrent genetic abnormality, were frequently mutated in more aggressive types of myeloid malignancies. Newly identified ancestral nature of PHF6 mutations specifically favor being followed by secondary driver RUNX1 mutations during leukemic evolution. Disclosures: Polprasert: MDS foundation: Research Funding. Maciejewski:Aplastic anemia&MDS International Foundation: Research Funding; NIH: Research Funding. Makishima:Scott Hamilton CARES grant: Research Funding; AA & MDS international foundation: Research Funding.

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